Many vaccines take the form of conjugates of an antigen associated with a carrier. For example, saccharides from bacteria have been used for many years in vaccines. As saccharides are T-independent antigens, however, they are poorly immunogenic. Conjugation to a carrier can convert T-independent antigens into T-dependent antigens, thereby enhancing memory responses and allowing protective immunity to develop. The most effective saccharide vaccines are therefore based on glycoconjugates, and the prototype conjugate vaccine was against Haemophilus influenzae type b (‘Hib’) [e.g. see chapter 14 of ref. [1]].
In assessing the immune response in a subject to a candidate conjugate vaccine, an assay may be used that detects the presence in a sample from the subject of an antibody raised in response to the conjugate. Such an assay usually comprises an immobilized substance that binds specifically to the antibody, for example, the antigen itself. Detection of bound antibody may then be achieved by use of, for example, chemical, biological or radioactive labelling. For example, detection of bound antibody may be achieved by use of a second antibody that recognizes antibodies from the subject, for example, where the assay is in an enzyme-linked immunosorbent assay (ELISA) format. Where the substance that binds specifically to the antibody is the antigen, a conjugate of the antigen associated with a protein may be used. The protein facilitates immobilization of the conjugate, for example to a multiwell plate. This facilitates detection of the antibody in the assay.
Use in the vaccine of a conjugate of the antigen associated with a protein can expose an artificial epitope that is a product of the conjugation. For example, such an artificial epitope may be present at the interface between the antigen and the protein. The candidate conjugate vaccine may therefore give rise to antibodies to this artificial epitope in addition to antibodies to the native antigen. Use in the assay of the conjugate of the antigen associated with a protein can also expose this artificial epitope, and as a result the assay will detect antibodies to this artificial epitope in addition to antibodies to the native antigen. This may give an inaccurate representation of the quantity of native antigen-specific antibodies in the sample being tested. Moreover, in some instances the immobilized conjugate can also cross-react with antibodies to other antigens [2].
In order to avoid detection of the artificial epitope, it is possible to simply use a physical mixture of the antigen and a protein [2]. However, where the antigen is hydrophilic, such as, for example, a saccharide, it can be difficult to immobilize the free antigen, for example to a plate. As a result, assays involving free antigens, either in physical mixture with a protein or alone, tend to have an inadequate level of sensitivity [3].
Development of a selective and sensitive method for detecting the presence of antibodies in a sample has been particularly problematic in the case of group B streptococcus (GBS; Streptococcus agalactiae). For example, in attempting to detect the presence of antibodies to a saccharide conjugate vaccine, some researchers have investigated an ELISA technique where the GBS saccharide is covalently linked to human serum albumin (HSA). The HSA component is intended to allow the conjugate to effectively bind to the ELISA plate and confer sufficient sensitivity on the assay [4].
However, other researchers have found that use of a conjugate in the assay exposes an artificial epitope (also present within the conjugate vaccine component) which is a product of the conjugation. The ELISA is therefore thought to detect antibodies to this epitope that are not antibodies to the native saccharide. The conjugate used in the assay can also cross-react with antibodies to pneumococcal saccharides [2]. Such a method is therefore insufficiently specific for antibodies to the GBS saccharide. In attempting to solve this problem, researchers have used a mixture of HSA and saccharide. However, this adversely affects the immobilization of the saccharide to the multiwell plate and the resulting assay is therefore insufficiently sensitive [3].
GBS infection constitutes a serious health threat to humans and animals. Of particular concern is the occurrence of GBS infections at the time of childbirth. Expectant mothers who are carriers of this bacterium are exposed to a risk of postpartum infection, and they may also transfer the infection to their child as the child passes through the birth canal. It is therefore useful to be able to determine whether maternal antibody levels are sufficient to protect against neonatal infection. Methods are therefore needed not only in the testing of a candidate conjugate vaccine but also in the surveillance of the effects of a vaccine in a patient, and in testing subjects for existing antibody levels to identify those subjects that may require vaccination. GBS infection is also a health threat to animals. For example, it is the cause of mastitis in dairy herds. Methods are therefore needed for testing and surveillance in veterinary contexts.
With the development of conjugate vaccines, and in particular conjugate vaccines based on GBS capsular saccharides, there is also a need for methods of assessing whether each batch of vaccine manufactured by a given process meets regulatory requirements, e.g. in terms of potency (e.g. immunogenicity). If the vaccine batch meets these requirements, then the vaccine should provide the expected immune response; ideally, there would be no need for further tests such as immunizing a test animal with a sample from the batch. Moreover, the requirements should be a more reliable assessment of potency than a physical characterization of the vaccine alone. The regulatory requirements for release of the vaccine are typically set by agencies such as the U.S. Food and Drug Administration (the FDA) in the United States and the European Medicines Agency (the EMEA) in Europe. It is therefore a further object of the invention to provide methods for assessing the potency of conjugate vaccines that meet these standards.
Accordingly, there remains a need for further and better methods for detecting the presence of antibodies in a sample, specifically of antibodies to a conjugate of an antigen associated with a carrier.